WO2006133107A2 - Sites de liaison specifiques dans le collagene pour integrines et utilisation de ceux-ci - Google Patents

Sites de liaison specifiques dans le collagene pour integrines et utilisation de ceux-ci Download PDF

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WO2006133107A2
WO2006133107A2 PCT/US2006/021769 US2006021769W WO2006133107A2 WO 2006133107 A2 WO2006133107 A2 WO 2006133107A2 US 2006021769 W US2006021769 W US 2006021769W WO 2006133107 A2 WO2006133107 A2 WO 2006133107A2
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collagen
seq
binding
integrin
peptide
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PCT/US2006/021769
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WO2006133107A3 (fr
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Magnus Hook
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The Texas A & M University System
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Priority to EP06772177A priority Critical patent/EP1889062A4/fr
Priority to AU2006255140A priority patent/AU2006255140A1/en
Priority to CA002620465A priority patent/CA2620465A1/fr
Publication of WO2006133107A2 publication Critical patent/WO2006133107A2/fr
Publication of WO2006133107A3 publication Critical patent/WO2006133107A3/fr
Priority to US13/346,565 priority patent/US20120208768A1/en
Priority to US13/770,852 priority patent/US10358464B2/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/566—Immunoassay; Biospecific binding assay; Materials therefor using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6887—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids from muscle, cartilage or connective tissue
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70546—Integrin superfamily, e.g. VLAs, leuCAM, GPIIb/GPIIIa, LPAM
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/02—Screening involving studying the effect of compounds C on the interaction between interacting molecules A and B (e.g. A = enzyme and B = substrate for A, or A = receptor and B = ligand for the receptor)

Definitions

  • the present invention relates generally to the field of computer- aided molecular modeling and interaction of extracellular matrix protein with receptors and cell signaling. More specifically, the present invention relates to identification of motifs within the extracellular matrix protein, collagen type III that binds I domains of the integrins and designing of specific inhibitors that inhibit the interaction of the I domain of integrin with the collagen.
  • Collagen is a major component of the extracellular matrix (ECM). At least 27 genetically different collagen types have been identified, each containing at least one dominant collagenous domain (Ramchandran, G. N., 1988). These collagenous domains have a characteristic triple helix structure formed by repeating GIy-X-Y sequences in each particiapting chain where X often is Proline and Y is hydroxyproline.
  • the collagen monomers often assemble into more complex structures of varying organizations such as fibrils (types NII 1 V and Xl), networks (types IV, VIII and X) and beaded filaments (type Vl) (Hulmes, D.J., 1992).
  • the fibrillar collagen types I and III are the major structural components of the ECM of skin, cardiac and vascular tissues, whereas type Il colllagen is a major structural component of cartilage. In addition to contributing to the structural integrity of the tissues, collagens also affect cell behaviour through interactions with other matrix proteins and cellular receptors (Prockop, D.J. and Kivirikko, 1995; Kuivaniemi, H., et al., 1997; Gelse, K. et. al., 2003; Myllyharju, J.and Kivirikko, K.I., 2001).
  • the integrins are a family of heterodimeric cell surface receptors involved in cell-cell and cell-substrate adhesion. They act as bridging molecules that link intracellular signaling molecules to the ECM through bidirectional signaling and control cell behaviour and tissue architecture (Hynes, R.O., 1992). Four integrins, ⁇ -i ⁇ i, «2 ⁇ i, ⁇ -io ⁇ i and ⁇ n ⁇ -ihave been shown to bind collagens (Kramer, R.H. and Marks, N. , 1989; Camper, L. et al., 1998; Veiling, T. et al., 1999).
  • Collagen binding by the four integrins is mediated by a ⁇ 200 amino acids long so-called inserted domain (I domain) found between blades 2 and 3 of the ⁇ -propeller domain of the a chains. All four I domains (ail, « 2 !, ⁇ -iol, etui) contain a metal ion-dependent adhesion site (MIDAS) that is required for coordinating a divalent cation and is essential for collagen binding.
  • MIDAS metal ion-dependent adhesion site
  • Synthetic collagen peptides containing the type I collagen derived sequences, GFOGER (SEQ ID NO: 1) or GLOGER (SEQ ID NO: 2) have been reported to bind with high affinity to ⁇ -il, C1 2 I and ⁇ nl; furthermore, synthetic peptides containing these sequences inhibit the binding of I domains to intact collagens (Knight, CG. et al., 1998; Zhang, W.M. et al., 2003; Siljander, P. R. et al., 2004).
  • the crystal structures of apo- ⁇ 2 l and CC2I in complex with a collagen peptide containing the GFOGER (SEQ ID NO: 1) sequence have been solved (Emsley, J.
  • GASGER SEQ ID NO: 3 was also reported to be recognized by the I domains but bound with lower affinity than GFOGER (SEQ ID NO: 1) and GLOGER (SEQ ID NO: 2) (Zhang, W.M. et al., 2003; Siljander, P. R. et al., 2004; Xu, Y. et al., 2000). Therefore, GFOGER (SEQ ID NO: 1) and GLOGER (SEQ ID NO: 2) are the only two known collagen-derived sequence motifs that support high affinity binding by the collagen-binding I domains.
  • GFOGER SEQ ID NO: 1
  • GLOGER SEQ ID NO: 2
  • CM I and ⁇ 2 l could bind to this collagen type (Nykvist, P. et al., 2000)
  • the present invention is directed to a binding motif for collagen binding I domains comprising amino acid sequence GROGER (SEQ ID NO: 4).
  • the present invention is further directed to a method of identifying an inhibitor of integrin-collagen interaction.
  • Such a method comprises designing a test compound comprising a sequence that is specifically recognized by the I domain of the integrin.wherein said design is based on computer-aided molecular modeling. Further, the level of binding of the integrin to the collagen is compared in the presence or absence of the test compound, where a decrease in binding of the integrin to the collagen in presence of the test compound indicates that the test compound is the inhibitor of the integrin- collagen interaction.
  • Figures 1A-C show the analyses of the binding of ⁇ -il and C1 2 I to fibrillar collagen type I -III by SPR. Representative profiles of the relative SPR responses of the binding of 1 ⁇ M CM I and az ⁇ to immobilized type I procollagen is shown in Figure 1A, type Il mature collagen is shown in Figure 1B and type III procollagen is shown in Figure 1C in the presence of 1 mM MgCI2. Responses to blank cells were subtracted. Y axis values are RUs normalized to the maximum RUs of all binding to type I - III collagens, respectively
  • Figure 2 shows electron micrographs of human type III procollagen in complex with ail or ⁇ 2 l following rotary shadowing.
  • the C- terminal propeptide of type III collagen indicated by arrowheads, appeared as a knob at one end of each of the collagen molecules.
  • the scale bars indicate 100 nm.
  • Figure 3 shows histograms of the binding events of ail and a 2 ⁇ along human type III procollagen. The events were binned every 10 nm, and the percentages of the binding events in each 10 nm bin over the total binding events counted are given. For CMI, a total of 269 binding events were counted, and for 0 2 I 1 a total of 299 events were counted.
  • Figures 4A-C show collagen peptides from putative high affinity binding sequences in type III collagen.
  • Figure 4A shows the amino acid sequence (SEQ ID NO: 18) of the high affinity binding region corresponding to 270 - 300 nm from the C-terminal propeptide, indicated by underlined letters, and its flanking area in human type III collagen.
  • FIG. 4B shows the amino acid sequences of the synthetic collagen peptides. Human type III collagen specific sequences are indicated by underlined and upper case letters, and type I collagen specific sequences are indicated by upper case letters. Three GPO triplets are included on either side to ensure the formation of triple helices.
  • Figure 4C shows circular dichroism spectra of the synthetic collagen peptides. Peptides #1, #2 and #3 in figure 4B and 4C are identified as SEQ ID NO: 15, 16 and 17, respectively.
  • Figures 5A-D show inhibition of the binding of ⁇ -il and ⁇ 2 l to collagens by synthetic collagen peptides.
  • Different concentrations of peptides (0.01-100 ⁇ M) were mixed with 0.5 ⁇ M CMI ( Figures 5A, 5C) and 5 ⁇ M C12I ( Figures 5B, 5D) before being added to microtiter wells coated with type III procollagen ( Figures 5A, 5B) or type I collagen ( Figures 5C, 5D).
  • Peptides #1 , #2 and #3 in these figures are identified as SEQ ID NO: 15, 16 and 17, respectively.
  • Figures 6A-C show representative SPR sensorgrams of ail and (X 2 I binding to immobilized peptide #1 (SEQ ID NO: 15). Increasing concentrations (0.5, 1 , 3, 6, 10 and 30 nM) of ail ( Figure 6A) or ⁇ 2 l ( Figure 6B), were passed over a surface containing synthetic peptide #1 (SEQ ID NO: 15). Responses to blank cells were subtracted. The binding was abolished in the presence of 2 mM EDTA ( Figure 6C).
  • Figure 7 shows adhesion of MRC-5 cells to synthetic collagen peptides.
  • Microtiter wells were coated with increasing concentrations of type III collagen or collagen peptides (SEQ ID NO: 15 or SEQ ID NO: 16). Both peptides were present in a triple helix conformation and immobilized to similar extent in the wells as tested by the amount of recombinant CAN bound to the
  • Figures 8A-D show analyses of GROGER (SEQ ID NO: 4) as the minimal binding sequence for Ct 1 1 and ⁇ 2 l.
  • Figure 8A shows the amino acid sequences of the synthetic collagen peptides (SEQ ID NOs: 19, 20, 21 , 15). Three GPO triplets are included on either side to ensure the formation of triple helices.
  • Figure 8B shows temperature-dependent denaturation profiles of synthetic collagen peptides. Ellipticity at 225 nm was measured as temperature increased from 10 to 50 0 C at the rate of 20°C/hr. Melting points of peptides were calculated by nonlinear fitting using the Boltzmann sigmoidal equation (GraphPad Prism).
  • Figures 8C and 8D show inhibition of the binding of ⁇ -il and cc 2 l to type III collagen by synthetic collagen peptides.
  • Figures 9A-D show computer modeling of the interactions between ⁇ 2 l and synthetic collagen peptide #1 (SEQ ID NO: 15).
  • the trailing and middle strands of the collagen peptide and the backbones of ⁇ 2 l are presented in green, yellow, and grey, respectively. All residues displayed are shown with oxygen in red, nitrogen in blue and carbon in white, green or yellow for residues from ⁇ 2 l, the trailing and the middle strand of collagen, respectively.
  • Figures 10A-D are computer modeling profiles showing specificity of collagen sequence to different I domains of collagen binding integrin alpha subunits.
  • FIG. 1OA shows the alpha-1 I domain in complex with collagen peptide.
  • Figure 1OB shows the alpha-2 I domain in complex with collagen peptide.
  • Figure 1OC shows alpha-10 I domain in complex with collagen peptide.
  • Figure 1OD shows alpha-11 I domain in complex with collagen peptide.
  • binding affinities is shown as follows: + represents binding affinity close to the template (blue), ++ represents slightly higher binding affinity (green), +++ represents much higher binding affinity (dark green), - represents lower binding affinity (yellow) and - - represents disrupted binding affinity (red).
  • F8 on middle and trailing strand were substituted simultaneously, E11 on middle strand was replaced for each mutation simulation and R12 on middle and trailing strand were substituted simultaneously for each mutation simulation.
  • the present invention identified a novel sequence motif, GROGER (SEQ ID NO: 4) from human type III collagen and characterized its binding to the I domains of integrins ⁇ i and 0C 2 .
  • GROGER SEQ ID NO: 4
  • the binding of recombinant I domains from integrins ⁇ i and ⁇ X 2 ( ⁇ -il and ⁇ 2l) to fibrillar collagens types l-lll was characterized and it was observed that each I domain bound to the three types of collagen with similar affinities.
  • the present invention also disclosed the use of computer- aided molecular modeling to identify sequences that are specifically recognized by individual I domains on the integrins. Thus, the finding of the present invention helps to understand the molecular interactions between collagens and integrins. It is further contemplated to synthesize peptides comprising the specific sequences identified by the modeling approach and test their utility in inhibiting integrin-collagen interaction and affect the biological and pathological conditions that arise due to such interaction.
  • Type III collagen is a homotrimeric molecule and is a member of the fibrillar collagen family. It co-localizes with type I collagen in tissues such as blood vessels and skin and plays a role in the development of these tissues (Prockop and Kivirikko, 1995; Liu, X et al ., 1997). In vitro, it has been reported that the type III collagen was able to support adhesion and spreading of cells expressing integrin ⁇ i ⁇ i or ⁇ 2 ⁇ i (Nykvist, P. et al., 2000). However, human type III collagen does not contain the two previously known high affinity integrin-binding motifs, GFOGER (SEQ ID NO: 1) and GLOGER (SEQ Id NO: 2).
  • the present invention compared the binding of ail and (X2I to the three different types of fibrillar collagen (types I, Il and III).
  • SPR Surface plasmon resonance
  • a high affinity integrin- binding site was located by rotary shadowing of / domains in complex with type III procollagen and a synthetic collagen triple helix peptide containing the GROGER (SEQ ID NO: 4) sequence was shown to bind with high affinity to the / domains and could serve as a substrate for integrin-dependent cell adhesion.
  • Other / domain-binding sites in type III collagen indicated by the rotary shadowing experiment might represent low affinity sites.
  • GROGER SEQ ID NO: 4
  • GFOGER GROGER sequence identified in this invention
  • GROGER integrin I domains
  • GFOGER GFOGER
  • GLOGER SEQ ID NO: 2
  • GROGER a somewhat higher affinity than GFOGER (SEQ ID NO: 1) and GLOGER (SEQ ID NO: 2) for ⁇ x 2 l and slightly lower affinity for ⁇ -il.
  • Bullfrog Type I O 2 (I) 142 The present invention also contemplates using a computer- aided molecular modeling approach to identify sequences that are specifically recognized by individual I domains on the integrins and thus can be used as base for developing integrin specific inhibitors. Such sequences can also be used to design specific binding sites in recombinant collagen or collagen-like proteins and to synthesize peptides that could be used as inhibitors of integrin-collagen interaction.
  • a binding motif for collagen-binding I domains comprising an amino acid sequence GROGER (SEQ ID NO: 4).
  • the I domains of integrins bound by such a motif include but may not be limited to ⁇ i and cx 2 integrins.
  • the motif may comprise a charged amino acid in second position.
  • the charged amino acid may form a hydrogen bond with carbonyl background of gluatmine at position 215 in a ⁇ ⁇ .
  • the charged amino acid may be arginine.
  • the motif may be present on N-terminal of human type III collagen or in the CM and 0:2 chain of human type I collagen.
  • a recombinant collagen or collagen like protein comprising the binding motif described supra.
  • Such a recombinant collagen or collagen-like protein may have sequence of SEQ ID NO: 15 or SEQ ID NO: 21.
  • an expression vector there is provided an expression vector.
  • Such an expression vector may comprise a DNA sequence encoding the recombinant collagen or collagen-like protein described earlier.
  • a host cell comprising and expressing the expression vector described earlier.
  • a synthetic collagen or collagen-like peptide comprising the binding motif described supra.
  • such a peptide may have sequence of SEQ ID NO: 15 or SEQ ID NO: 21.
  • such a peptide has a triple helical structure.
  • such a synthetic peptide may bind I domains of integrins ⁇ i and ⁇ 2 .
  • a method of identifying an inhibitor of an integrin- collagen interaction comprising: designing a test compound comprising a sequence that is specifically recognized by the I domain of the integrin, where the design is based on computer-aided molecular modeling, and comparing the level of binding of the integrin to the collagen in the presence or absence of the test compound, where a decrease in binding of the integrin to the collagen in presence of the test compound indicates that the test compound is the inhibitor of the integrin-collagen interaction.
  • the examples of integrin that are bound by such inhibitors may not be limited to but include ⁇ i ⁇ i, ⁇ 2 ⁇ i, ⁇ io ⁇ i or ⁇ n ⁇ i.
  • the collagen whose interaction with the integrin is affected may be a type I 1 Il or III collagen.
  • the inhibitor has a triple helical structure and may comprise an amino acid sequence including but not limited to GFPGER (SEQ ID NO: 6), GFOGEN (SEQ ID NO: 7), GFOGEK (SEQ ID NO: 8), GNOGER (SEQ ID NO: 9), GSOGER (SEQ ID NO: 10), GVOGER (SEQ ID NO: 11) or GPOGER (SEQ ID NO: 12).
  • an inhibitory compound that is identified by the method described above.
  • a recombinant collagen or collagen-like protein comprising the inhibitory sequence indentified by the method described supra.
  • an expression vector comprising a DNA sequence encoding the recombinant collagen or collagen-like protein described supra.
  • a host cell comprising and expressing the expression vector described earlier.
  • a synthetic collagen or collagen-like peptide comprising the inhibitory sequence identified by the method described supra.
  • the inhibitory sequence in a triple helical structure may not be limited to but includes GFPGER (SEQ ID NO: 6), GFOGEN (SEQ ID NO; 7), GFOGEK (SEQ ID NO: 8), GNOGER (SEQ ID NO: 9), GSOGER (SEQ ID NO: 10), GVOGER (SEQ ID NO: 11) or GPOGER (SEQ ID NO: 12).
  • GFPGER SEQ ID NO: 6
  • GFOGEN SEQ ID NO; 7
  • GFOGEK SEQ ID NO: 8
  • GNOGER SEQ ID NO: 9
  • GSOGER SEQ ID NO: 10
  • GVOGER SEQ ID NO: 11
  • GPOGER SEQ ID NO: 12
  • a method of inhibiting integrin-collagen interaction comprising: contacting a sample comprising the integrin and the collagen with the peptide discussed supra, where the peptide binds the integrin with a greater affinity than the collagen, thereby inhibiting the integrin-collagen interaction.
  • biological processes contributed by integrin-collagen interaction and may be affected by the peptide may not be limited to but include cell adhesion, cell migration, cell proliferation, cell differentiation, angiogenesis, platelet aggregation or extracellular matrix assembly.
  • a pharmaceutical composition comprising the inhibitory compound identified by the method described supra and a pharmaceutically acceptable carrier.
  • a method of treating an individual having a pathophysiological condition resulting from integrin-collagen interaction comprising the step of administering to the individual a pharmacologically effective amount of the composition discussed supra, such that the composition inhibits the integrin- collagen interaction, thereby treating the individual having the pathophysiological condition.
  • the pathophysiological conditions am may not be limited to but include inflammation, tumor growth, metastasis or angiogenesis.
  • the term “compound” or “inhibitor” or “inhibitory compound” means a molecular entity of natural, semi-synthetic or synthetic origin that blocks, stops, inhibits, and/or suppresses integrin interactions with collagen.
  • the term “contacting” refers to any suitable method of bringing integrin, collagen and the inhibitory compound, as described, or a cell comprising the same and the inhibitory compound. In vitro or ex vivo this is achieved by exposing the integrin, collagen to the inhibitory compound or cells comprising the same to the compound or inhibitory agent in a suitable medium. For in vivo applications, any known method of administration is suitable as described herein.
  • Recombinant I domains of integrin ⁇ 1 and ⁇ 2 subunits were generated and isolated as (Xu 1 Y et al., 2000; Rich, R.L. et al., 1999). Purified recombinant proteins were examined by SDS-polyacrylamide gel electrophoresis (PAGE) followed by staining with Coomassie blue.
  • Frozen yeast cells expressing recombinant type I and III procollagens were obtained from FibroGen, lnc (San Francisco, CA). The yeast cells expressed both genes encoding human collagen and prolyl 4- hydroxylase enabling formation of hydroxyproline residues and thermally stable triple helical collagen. The cells were thawed in an ambient- temperature water bath and resuspended in a Start buffer (0.1 M Tris, 0.4 M NaCI 1 25 mM EDTA 1 1 mM phenylmethylsulfonyl fluoride (PMSF) 1 1 mM pepstatin, pH 7.5).
  • Start buffer 0.1 M Tris, 0.4 M NaCI 1 25 mM EDTA 1 1 mM phenylmethylsulfonyl fluoride (PMSF) 1 1 mM pepstatin, pH 7.5.
  • the cells were lysed using a French press, and the lysate was centrifuged at 30,000 x g for 30 min at 4 0 C. The supernatant was filtered through a 0.45-mm membrane, and the pH of the filtrate was adjusted to 7.5.
  • An affinity column was prepared by coupling a recombinant collagen-binding MSCRAMM from Staphylococcus aureus (Patti et al., 1992) to CNBr-activated Sepharose 4B (Amersham Biosciences). The supernatant was applied to the column and incubated overnight at 4°C. The column was washed with the Start buffer and bound material was eluted with 0.5 M acetic acid.
  • Fractions were examined by SDS-PAGE (4%/8%) under reducing conditions followed by Coomassie blue staining. Fractions with procollagen were pooled. The concentration of the procollagen was estimated by comparing its band intensity with that of known amount of type I collagen (Vitrogen) in Coomassie blue stained SDS-gels.
  • HBS buffer 25 mM HEPES, 150 mM NaCI, pH 7.4
  • 5 mM ⁇ -mercaptoethanol, 1 mM MgCI2, and 0.05% octyl-D-glucopyranoside were passed over the immobilized collagen at 30 ⁇ l/min for 4 min. Regeneration of the collagen surfaces was achieved with 20 ⁇ l of HBS containing 0.01% SDS.
  • SPR sensorgrams from different injections were overlaid using the BIAevaluation software (BIAcore AB). Data from the steady state portion of the sensorgrams were used to determine the binding affinities.
  • values for the binding ratio, Vbound, and the concentration of free protein, [P]f re e were calculated using the equations described previously (Rich, R. L. et al., 1999).
  • Microtiter wells (Immulon 4, Thermo Labsystems) were coated with 1 ⁇ g of mature bovine type I collagen (Vitrogen) or purified human type III procollagen in HBS for 2 hrs at room temperature. The wells were washed with HBS and incubated with a blocking buffer (HBS containing 0.1% w/v ovalbumin and 0.05% v/v Tween 20) overnight at 4 0 C. Varying concentrations of peptides were mixed with fixed concentrations of recombinant I domains in the blocking buffer containing 1 mM MgCI2 and 5 mM ⁇ -mercaptoethanol and then added to the wells.
  • a blocking buffer HBS containing 0.1% w/v ovalbumin and 0.05% v/v Tween 20
  • Bound antibodies were quantified by adding 100 ⁇ l of 1.3 M diethanolamine, pH 9.8, containing 1 mM MgCb, and 1 mg/ml p-nitrophenyl phosphate (Southern Biotechnology Associates, Birmingham, AL) to each well. The absorbance at 405nm (/1405 nm) was measured after 20 -40 min of incubation at room temperature. Background binding to the wells was determined by incubating the I domains in wells that had been treated with blocking buffer alone. These values were subtracted from the values generated in the collagen-coated wells to determine collagen-specific binding.
  • Peptides were synthesized by a solid phase method on a TentaGel R RAM resin (RAPP Polymere GmbH, Tubingen, Germany) using Fmoc chemistry and a model 396 MBS Multiple Peptide Synthesizer from Advanced ChemTech Inc. (Louisville, KY). Fmoc amino acids were purchased from Novabiochem, San Diego, CA. Coupling of amino acids was carried out twice using diisopropylcarbodiimide/1-hydroxybenzotriazole for 60 min.
  • Fmoc deprotection was carried out using a mixture of 2% (v/v) piperidine and 2% (v/v) 1,8-diazabicyclo-[5.4.0]undec-7-ene in dimethylformamide followed by treatment with 25% piperidine in dimethylformamide. Side chains were protected with the following groups: f-butyl (GIu, Ser, and hydroxy-Pro), 2,2,5,7,8-pentamethylchroman-6-sulfonyl (Arg), and trityl (GIn).
  • peptide resins were washed thoroughly with dimethylformamide, ethanol, and ether and then dried in a vacuum desiccator. Peptides were released from the resin by treatment with a mixture of trifluoroacetic acid, thioanisole, ethanedithiol, and triethylsilane (90:5:2.5:2.5 by volume) for 8 h. The resins were filtered, and the peptides were precipitated with cold anhydrous ether. The precipitate was washed with anhydrous ether three times and dried. The cleaved peptides were analyzed by reverse phase high pressure liquid chromatography on a Waters 625 liquid chromatography system (Milford, MA) using a Waters Delta-Pak C18 column. EXAMPLE 7
  • Synthetic collagen peptides were analyzed by CD spectroscopy, as described previously (Xu, Y. et al., 2000) with the following modifications. Briefly, peptides were dissolved in HBS to a concentration of 50 ⁇ M. CD spectra were collected on a Jasco J720 spectropolarimeter (Tokyo, Japan) from 190 to 240 nm, with bandwidth of 1 nm and integrated for 1 s at 0.2 nm intervals. Samples were measured at room temperature using cuvettes with 0.02 cm path length. For temperature-dependant denaturation analysis, 30 mM of peptides were added to a thermostatically controlled cuvette with a 0.5 cm path length used. Thermal transition profiles were recorded at 225 nm as described above with a temperature slope of 20°C/hr. To calculate the temperature melting points, the thermal transition profiles were fitted with
  • the human recombinant mature type III collagen used for cell attachment assays was purchased from FibroGen. All cell culture media components were purchased from Invitrogen.
  • the human lung fibroblast cell line MRC-5 was purchased from American Type Culture Collection (ATCC) (Manassas, VA). The cells were cultured and passaged in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 100 unit/ml penicillin and 100 ⁇ g/ml streptomycin. The cells were grown to subconfluence and passaged every 2-3 days.
  • DMEM Dulbecco's modified Eagle's medium
  • MRC-5 cells were starved overnight in serum deficient DMEM containing penicillin and streptomycin, then detached using 1 mM EDTA and 0.025% trypsin at 37°C for 2 minutes. The cells were washed with PBS and resuspended in DMEM containing 0.2% BSA supplemented with 2 mM
  • the coordinates of the crystal structure of 012I in complex with a synthetic collagen peptide were obtained from Protein Data Bank (code 1dzi) and used as a template for the model studies. First, the Phe residues in both the middle and trailing strands were replaced by Arg residues. Then local minimization was carried out in sizes of 5 A for best fit. Several basic components (i.e., hydrogen bond, van der Waals and electrostatic interactions) contributing to the binding energy between 0: 2 ! and the mutated collagen peptide were analyzed. The molecular modeling experiment was carried out under ECEPP/3 force field by using the ICM software (Molsoft, La JoIIa, CA).
  • the BIAcore 3000 system used in the present invention had higher sensitivity of detection which enabled examination of the interactions between 01 2 I and collagens at a sub-micromolar concentration range.
  • the dissociation constants (KD) of these interactions are summarized in Table 2.
  • the binding affinities of « 2 ! to the three types of collagen appeared to be slightly more variable.
  • the KD values for the high affinity binding class range from ⁇ 0.3 ⁇ M for types I and III collagen, to 1.75 ⁇ M for type Il collagen, whereas the KD values for the low affinity binding class range from ⁇ 4 ⁇ M for type I collagen, to ⁇ 16.5 ⁇ M and - 14.5 ⁇ M for type Il and III collagen, respectively.
  • the two recombinant I domains also exhibited different binding kinetics to the collagens as indicated by the shape of the corresponding SPR sensorgrams (Fig. 1).
  • the binding characteristics of the interactions between ⁇ -il/c ⁇ l and type III collagen are similar to those of the interactions between ⁇ il/ ⁇ 2 l and type l/ll collagen.
  • KD was calculated by equilibrium analysis. Data are presented as mean value ⁇ S. E of three independent studies.
  • GFOGER and GLOGER 1 Two sequence motifs, GFOGER and GLOGER 1 were identified as high affinity binding sites in triple helical collagen for ⁇ -il, ⁇ 2 l and ⁇ nl (Zhang, W.M. et al., 2003; Siljander, P.R. et al., 2004, Xu, Y et al., 2000). The fact that these sequences were present in type I and Il collagen but not in type III collagen suggested the presence of at least one novel high affinity binding site in type III collagen. To locate the high affinity binding region(s) in type III collagen, collagen and I domain complexes were examined by rotary shadowing followed by electron microscopy (EM). Type III procollagen was used in these experiments since it contains a globular-shaped C-terminal propeptide that allows the determination of the orientation of collagen molecules in EM.
  • EM electron microscopy
  • Type III procollagen was incubated with ail or ⁇ 2 l under binding conditions and the complexes were then subjected to rotary shadowing and EM.
  • the helical portion of the majority of the collagen molecules was found to be ⁇ 300 nm long, indicating that these molecules were mostly intact, full- length molecules.
  • Type III collagen is a homotrimer composed of three ⁇ 1(lll) polypeptides, each containing 1029 amino acid residues in the mature chain (GenBankTM accession number P02461). Given that the average collagen molecule measured 300 nm, the average length per residue of collagen was 0.29nm (3.43 amino acid residues/nm), which was consistent with previous calculations (Bella et al., 1994). Based on this correlation, the region located 270 - 300 nm region from C-terminal end of the mature chain corresponded to amino acid residues 168 - 270 of the ⁇ 1(lll) chain. This stretch of sequence contained one GER motif preceded by GROGRO (SEQ ID NO: 13) and followed by GLO (Fig. 4 A).
  • the three synthetic peptides were examined for their ability to form collagen-like triple helices by CD spectroscopy.
  • the CD spectra of all four peptides showed the characteristic ellipticity maxima at 220-225 nm, indicating that they were capable of forming collagen-like triple helices (Fig. 4C).
  • the temperature-dependent unfolding of the triple helix was followed by monitoring the CD at 225 nm.
  • the reduction of the maxima was seen from about 35°C-with melting points for the triple helix structure of these peptides recorded between 41 and 44 0 C (data for peptide #1 are shown in Fig 8B).
  • the data discussed herein showed that the peptides formed triple helix structure at temperatures (4-25 0 C) used in the following experiments.
  • type III collagen peptides contained high affinity binding sites for ail and CC2I, their ability to inhibit the binding of recombinant I domains to type I and III collagens was examined using ELISAs type assays.
  • Various concentrations of peptides (0.01-100 ⁇ M) were incubated with recombinant I domains before the mixtures were added to microtiter wells coated with type I or III collagen.
  • Peptide #2 (SEQ ID NO: 16) at 100 ⁇ M inhibited the binding of ⁇ -il and ⁇ 2 l to type III collagen by 40% and 60% respectively, suggesting that although peptide #2 (SEQ ID NO: 16) was recognized by the I domains, it was not a high affinity binding site.
  • the control peptide #3 (SEQ ID NO: 17) did not show any inhibitory activity.
  • the IC 50 values of peptide #1 (SEQ ID NO: 15) with ail and CX 2 I binding to type III collagen were 1.0 ⁇ 0.6 ⁇ M and 1.9 ⁇ 0.9 ⁇ M, respectively.
  • Peptide #1 (SEQ ID NO: 15) was immobilized onto a CM5 chip. Increasing concentrations of I domains (0.5 - 30 nM) were passed over the surface containing peptide #1 (SEQ ID NO: 15). ail and 01 2 I
  • GROGER (SEQ ID NO: 4) is a minimal ⁇ i IZa 7 I high affinity binding motif
  • peptide #1 SEQ ID NO: 15
  • a shorter peptide containing the GROGER (SEQ ID NO: 4) sequence flanked by three GPO repeats at either ends was synthesized.
  • Peptides containing GFOGER (SEQ ID NO: 1) and GLOGER (SEQ ID NO: 2) motifs were also made for comparison (Fig. 8A). All three peptide were able to form triple helices as shown by their ellipticity maxima around 225 nm in the CD spectra (data not shown). In addition, all three showed a sharp decrease in their ellipticity at 225 nm as temperature increased.
  • the melting temperature (Tm) of the GFOGER (SEQ ID NO: 1), GLOGER (SEQ ID NO: 2) and GROGER (SEQ ID NO: 4) peptides were determined to be between 37°C and 41 0 C.
  • the denaturation profile of GROGER (SEQ ID NO: 4) peptide was compared to that of the peptide #1 (SEQ ID NO: 15).
  • the Tm of the GROGER (SEQ ID NO: 4) peptide was 41 0 C, slightly lower than the Tm of peptide #1 (SEQ ID NO: 15) which was determined to 43°C (Fig. 8B).
  • GROGER SEQ ID NO: 4
  • GFOGER GFOGER
  • GLOGER SEQ ID NO: 2
  • GROGER SEQ ID NO: 4
  • IC 50 values of GFOGER SEQ ID NO: 1
  • GLOGER SEQ ID NO: 2
  • GROGER SEQ ID NO: 4

Abstract

L'invention concerne une séquence de liaison à affinité élevée dans le collagène de type III destinée à des domaines I d'intégrine de liaison du collagène. L'invention concerne des procédés utilisés pour caractériser la séquence, les peptides comprenant cette séquence et l'utilisation des peptides dans la réalisation de l'adhésion cellulaire. L'invention concerne, en outre, des procédés permettant d'identifier des inhibiteurs d'intégrine spécifiques, des séquences de ces inhibiteurs et l'utilisation de ceux-ci dans l'inhibition d'états pathophysiologiques pouvant apparaître en raison de l'interaction intégrine-collagène.
PCT/US2006/021769 2005-06-03 2006-06-05 Sites de liaison specifiques dans le collagene pour integrines et utilisation de ceux-ci WO2006133107A2 (fr)

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AU2006255140A AU2006255140A1 (en) 2005-06-03 2006-06-05 Specific binding sites in collagen for integrins and use thereof
CA002620465A CA2620465A1 (fr) 2005-06-03 2006-06-05 Sites de liaison specifiques dans le collagene pour integrines et utilisation de ceux-ci
US13/346,565 US20120208768A1 (en) 2005-06-03 2012-01-09 Specific binding sites in collagen for integrins and use thereof
US13/770,852 US10358464B2 (en) 2005-06-03 2013-02-19 Specific binding sites in collagen for integrins and use thereof

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WO2011008303A2 (fr) * 2009-07-17 2011-01-20 The Texas A & M University System Collagènes sur mesure et leurs utilisations
US9725498B2 (en) 2009-07-17 2017-08-08 The Texas A&M University System Designer collagens and use thereof
WO2013134269A2 (fr) * 2012-03-06 2013-09-12 Kci Licensing, Inc. Nouvelles compositions, leur préparation et leur utilisation
FR2997083B1 (fr) * 2012-10-19 2014-12-12 Nvh Medicinal Proteines recombinantes derivees du collagene a activite de liaison au facteur willebrand
WO2014123665A1 (fr) 2013-02-06 2014-08-14 Kci Licensing, Inc. Polymères, leur préparation et utilisation
WO2017020025A1 (fr) * 2015-07-29 2017-02-02 Kiick Kristi Conjugués biologiques thermosensibles et leur administration contrôlée de charges
US20180236141A1 (en) * 2017-02-17 2018-08-23 The Texas A&M University System Prokaryotic Collagen Therapeutics for Postoperative Adhesions
US11180541B2 (en) 2017-09-28 2021-11-23 Geltor, Inc. Recombinant collagen and elastin molecules and uses thereof
EP3953380A4 (fr) 2019-04-12 2023-01-25 Geltor, Inc. Élastine de recombinaison et production associée
MX2022009036A (es) 2020-01-24 2022-11-14 Geltor Inc Colageno dietetico no de animales.
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US8252553B2 (en) 2012-08-28
US20120208768A1 (en) 2012-08-16
EP1889062A2 (fr) 2008-02-20
US20090203627A1 (en) 2009-08-13
US10358464B2 (en) 2019-07-23
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US20130150300A1 (en) 2013-06-13
WO2006133107A3 (fr) 2009-04-30

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